Method for dispensing and determining a microvolume of sample liquid

ABSTRACT

A low volume liquid handling system is described which includes a microdispenser employing a piezoelectric transducer attached to a glass capillary, a positive displacement pump for priming and aspirating liquid into the microdispenser, controlling the pressure of the liquid system, and washing the microdispenser between liquid transfers, and a pressure sensor to measure the liquid system pressure and produce a corresponding electrical signal. The pressure signal is used to verify and quantify the microvolume dispensed and is used to perform automated calibration and diagnostics on the microdispenser.

This application is a divisional of 09/012,174 filed Jan. 22, 1998 whichis a continuation of 08/656,455 filed May 31, 1996 and now abandoned.

FIELD OF THE INVENTION

The present invention relates to an apparatus and process forcontrolling, dispensing and measuring small quantities of fluid. Morespecifically, the present invention senses pressure changes to ascertainand confirm fluid volume dispensed and proper system functioning.

BACKGROUND OF THE INVENTION

Advances in industries employing chemical and biological processes havecreated a need for the ability to accurately and automatically dispensesmall quantities of fluids containing chemically or biologically activesubstances for commercial or experimental use. Accuracy and precision inthe amount of fluid dispensed is important both from the standpoint ofcausing a desired reaction and minimizing the amount of materials used.

Equipment for dispensing microvolumes of liquid have been demonstratedwith technologies such as those developed for ink jet applications.However, ink jet equipment has the advantage of operating with aparticular ink (or set of inks) of known and essentially fixed viscosityand other physical properties. Thus, because the properties of the inkbeing used are known and fixed, automatic ink jet equipment can bedesigned for the particular ink specified. Direct use of ink jettechnology with fluids containing a particular chemical and biologicalsubstance of interest ("transfer liquid") is more problematic. Suchtransfer liquids have varying viscosity and other physical propertiesthat make accurate microvolume dispensing difficult. Automaticmicrovolume liquid handling systems should be capable of handling fluidsof varying viscosity and other properties to accommodate the wide rangeof substances they must dispense. Another aspect of this problem is theneed to accommodate accurately dispensing smaller and smaller amounts oftransfer liquid. Especially in the utilization and test of biologicalmaterials, it is desirable to reduce the amount of transfer liquiddispensed in order to save costs or more efficiently use a small amountof material available. It is often both desirable and difficult toaccurately dispense microvolumes of transfer liquid containingbiological materials. Knowing the amount of transfer liquid dispensed inevery ejection of transfer liquid would be advantageous to an automatedsystem.

Another difficulty with dispensing microvolumes of transfer liquidarises due to the small orifices, e.g., 20-80 micrometers in diameter,employed to expel a transfer liquid. These small orifice sizes aresusceptible to clogging. Further exacerbating the clogging problem arethe properties of the substances sometimes used in the transfer liquid.Clogging of transfer liquid substances at the orifice they are expelledfrom, or in other parts of the dispenser, can halt dispensing operationsor make them far less precise. Therefore, it would be desirable to beable to detect when such conditions are occurring, and to be able toautomatically recover from these conditions. Failure of a microvolumedispenser to properly dispense transfer fluid can also be caused byother factors, such as air or other compressible gases being in thedispensing unit. It would be desirable to detect and indicate when amicrovolume dispenser is either not dispensing at all, or not dispensingthe desired microvolume ("misfiring").

In order to achieve an automated microvolume dispensing system it wouldbe desirable to ensure in realtime that the transfer liquid is withinsome given range of relevant system parameters in order to rapidly andaccurately dispense transfer liquid droplets of substantially uniformsize. Because industry requires rapid dispensing of microvolume amountsof transfer liquid, it is desirable to be able to ascertain transferliquid volume dispensed, and to be able to detect and recover fromdispensing problems in realtime.

SUMMARY OF THE INVENTION

It is a primary object of the present invention to provide a microvolumeliquid handling system which is capable of accurately verifyingmicrovolume amounts of transfer liquid dispensed by sensing acorresponding change in pressure in the microvolume liquid handlingsystem.

It is also an object of the present invention to provide a microvolumeliquid handling system which can accurately measure an amount ofdispensed liquid regardless of transfer liquid properties such asviscosity.

It is another object of the present invention to provide a microvolumeliquid handling system which can transfer microvolume quantities offluids containing chemically or biologically active substances.

It is still another object of the present invention to provide amicrovolume liquid handling system which senses pressure changesassociated with clogging and misfiring to indicate such improperoperation.

It is yet another object of the present invention to provide amicrovolume liquid handling system which can verify that the transferliquid is maintained within a given range of negative pressure (withrespect to ambient atmospheric pressure) in order to accurately dispensemicrovolume amounts of transfer liquid and optimize the operation of themicrodispenser.

Other objects and advantages of the present invention will be apparentfrom the following detailed description.

Accordingly, the foregoing objectives are realized by providing amicrovolume liquid handling system which includes a positivedisplacement pump operated by a stepper motor, a piezoresistive pressuresensor, and an electrically controlled microdispenser that utilizes apiezoelectric transducer bonded to a glass capillary. The microdispenseris capable of rapidly and accurately dispensing sub-nanoliter ("nl")sized droplets by forcibly ejecting the droplets from a small nozzle.

The present invention includes a system liquid and a transfer liquid inthe dispensing system separated by a known volume of air ("air gap")which facilitates measuring small changes in pressure in the systemliquid that correlate to the volume of transfer liquid dispensed. Thetransfer liquid contains the substances being dispensed, while in onepreferred embodiment the system liquid is deionized water. Each time adroplet in the microvolume dispensing range is dispensed, the transferliquid will return to its prior position inside the microdispenserbecause of capillary forces, and the air gap's specific volume will beincreased corresponding to the amount of transfer liquid dispensed. Thishas the effect of decreasing pressure in the system liquid line which ismeasured with a highly sensitive piezoresistive pressure sensor. Thepressure sensor transmits an electric signal to control circuitry whichconverts the electric signal into a digital form and generates anindication of the corresponding volume of transfer liquid dispensed. Anadvantage of the present invention is its insensitivity to the viscosityof the transfer liquid. This is because the pressure change in thesystem liquid corresponds to the microvolume dispensed, without beingdependent on the dispensed fluid viscosity. The present inventionpossesses unique capabilities in microvolume liquid handling. Thissystem is capable of automatically sensing liquid surfaces, aspiratingliquid to be transferred, and then dispensing small quantities of liquidwith high accuracy, speed and precision. The dispensing is accomplishedwithout the dispenser contacting the destination vessel or contents. Afeature of the present invention is the capability to positively verifythe microvolume of liquid that has been dispensed during realtimeoperation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of the a microvolume liquid handling systemembodying the present invention;

FIG. 2 is a schematic of a positive displacement pump;

FIG. 3 is an illustration of a microdispenser and a piezoelectrictransducer; and

FIG. 4 is a graph depicting the system line pressure during amicrodispenser dispense.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and will herein be described in detail. Itshould be understood, however, that it is not intended to limit theinvention to the particular forms disclosed. On the contrary, theintention is to cover all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theappended claims.

DETAILED DESCRIPTION OF THE INVENTION

Turning now to the drawings and referring first to FIG. 1, a microvolumeliquid handling system 10 is illustrated. The microvolume liquidhandling system 10 includes a positive displacement pump 12, a pressuresensor 14 and a microdispenser 16. Tubing 18 connects the positivedisplacement pump 12 to the pressure sensor 14 and the pressure sensor14 to the microdispenser 16. The positive displacement pump 12 moves asystem liquid 20 through the pressure sensor 14 and the microdispenser16. After the system 10 is loaded with system liquid 20, an air gap 22of known volume, then an amount of transfer liquid 24, are drawn intothe microdispenser 16 in a manner described below. The transfer liquid24 contains one or more biologically or chemically active substances ofinterest. In one preferred embodiment the microdispenser 16 expels (orsynonymously, "shoots") sub-nanoliter size individual droplets 26 whichare very reproducible. The expelled droplets 26 of transfer liquid 24are on the order of 0.45 nanoliters per droplet 26 in one preferredembodiment, but they can be as small as 5 picoliters. For example, ifone desires to expel a total of 9 nanoliters of transfer liquid 24, thenthe microdispenser 16 will be directed to expel 20 droplets 26. Droplet26 size can be varied by varying the magnitude and duration of theelectrical signal applied to the microdispenser 16. Other factorsaffecting droplet size include: the size of the nozzle opening at thebottom of the microdispenser, the pressure at the microdispenser inlet,and properties of the transfer liquid.

Referring now to FIGS. 1 and 2, in one preferred embodiment the positivedisplacement pump 12 is a XL 3000 Modular Digital Pump manufactured byCavro Scientific Instruments, Inc., 242 Humboldt Court, Sunnyvale,Calif. 94089. The positive displacement pump 12 includes stepper motor28 and stepper motor 29, and a syringe 30. The syringe 30 includes aborosilicate glass tube 32 and a plunger 34 which is mechanicallycoupled through a series of gears and a belt (not shown) to the steppermotor 28. Stepper motor 28 motion causes the plunger 34 to move up ordown by a specified number of discrete steps inside the glass tube 32.The plunger 34 forms a fluidtight seal with the glass tube 32. In onepreferred embodiment syringe 30 has a usable capacity of 250 microliterswhich is the amount of system liquid 20 the plunger 34 can displace inone full stroke. Depending on the selected mode of operation, thestepper motor 28 is capable of making 3,000 or 12,000 discrete steps perplunger 34 full stroke. In one preferred embodiment the stepper motor 28is directed to make 12,000 steps per fill plunger 34 stroke with eachstep displacing approximately 20.83 nanoliters of system liquid 20. Inone preferred embodiment the system liquid 20 utilized is deionizedwater.

Digitally encoded commands cause the stepper motor 28 within thepositive displacement pump 12 to aspirate discrete volumes of liquidinto the microdispenser 16, wash the microdispenser 16 between liquidtransfers, and to control the pressure in the system liquid 20 line formicrovolume liquid handling system 10 operation. The positivedisplacement pump 12 is also used to prime the system 10 with systemliquid 20 and to dispense higher volumes of liquid through themicrodispenser 16, allowing dilute solutions to be made. The positivedisplacement pump 12 can also work directly with transfer liquid 24.Thus, if desired, transfer liquid 24 can be used as system liquid 20throughout the microvolume liquid handling system 10.

To prime the microvolume liquid handling system 10, the control logic 42first directs a 3-axis robotic system 58 through electrical wire 56 toposition the microdispenser 16 over a wash station contained on therobotic system 58. In one preferred embodiment the microvolume liquidhandling system 10 includes, and is mounted on, a 3-axis robotic systemis a MultiPROBE CR10100, manufactured by Packard Instrument Company,Downers Grove, Ill. The positive displacement pump 12 includes a valve38 for connecting a system liquid reservoir 40 to the syringe 30. Aninitialization control signal is transmitted through the electricalcable 36 to the pump 12 by control logic 42 which causes the valve 38 torotate connecting the syringe 30 with the system fluid reservoir 40. Thecontrol signal also causes the stepper motor 28 to move the plunger 34to its maximum extent up (Position 1 in FIG. 2) into the borosilicateglass tube 32. The next command from the control logic 42 causes thestepper motor 28 to move the plunger 34 to its maximum extent down(Position 2 in FIG. 2) inside the tube 32, to extract system liquid 20from the system reservoir 40. Another command from the control logic 42directs the valve 38 to rotate again, causing the syringe 30 to beconnected with the tubing 18 connected to the pressure sensor 14. In onepreferred embodiment the tubing 18 employed in the microvolume liquidhandling system 10 is Natural Color Teflon Tubing made by ZeusIndustrial Products, Inc., Raritan, N.J., with an inner diameter of0.059 inches and an outer diameter of 0.098 inches. The next commandfrom the control logic 42 to the positive displacement pump 12 causesthe system liquid 20 inside of the syringe 30 to be pushed into themicrovolume liquid handling system 10 towards the pressure sensor 14.Because the microvolume liquid handling system 10 typically requiresabout 4 milliliters of system fluid to be primed, the sequence of stepsdescribed above must be repeated about 16 times in order to completelyprime the microvolume liquid handling system 10.

The control logic 42 receives signals from the pressure sensor 14through an electrical line 46. The signals are converted from an analogform into a digital form by an A/D (analog to digital) converter 44 andused by the control logic 42 for processing and analysis. In onepreferred embodiment the A/D conversion is a PC-LPM-16 Multifinction I/OBoard manufactured by National Instruments Corporation, Austin, Texas.At various points in the liquid transfer process described herein, thecontrol logic 42 receives signals from the pressure transducer 14, andsends command signals to the pump 12, microdispenser electronics 51, andthe 3-axis robotic system 58. Within the control logic 42 are theencoded algorithms that sequence the hardware (robotic system 58, pump12, and microdispenser electronics 51) for specified liquid transferprotocols as described herein. Also within the control logic 42 are theencoded algorithms that process the measured pressure signals to: verifyand quantify microdispenses, perform diagnostics on the state of themicrovolume liquid handling system, and automatically perform acalibration of the microdispenser for any selected transfer liquid 24.

The pressure sensor 14 senses fluctuations in pressure associated withpriming the microvolume liquid handling system 10, aspirating transferliquid 24 with pump 12, dispensing droplets 26 with microdispenser 16,and washing of microdispenser 16 using pump 12. In one preferredembodiment the pressure sensor 14 is a piezoresistive pressure sensorpart number 26PCDFG6G, from Microswitch, Inc., a Division of Honeywell,Inc., 11 West Spring Street, Freeport, Ill. 61032. Also included withthe pressure sensor 14 in the block diagram in FIG. 1 is electricalcircuitry to amplify the analog pressure signal from the pressuresensor. The pressure sensor 14 converts pressure into electrical signalswhich are driven to the A/D converter 44 and then used by the controllogic 42. For example, when the microvolume liquid handling system 10 isbeing primed, the pressure sensor 14 will send electrical signals whichwill be analyzed by the control logic 42 to determine whether theyindicate any problems within the system such as partial or completeblockage in the microdispenser 16.

Once the microvolume liquid handling system 10 is primed, the controllogic 42 sends a signal through electrical wire 56 which instructs therobotic system 58 to position the microdispenser 16 in air over thetransfer liquid 24. The control logic 42 instructs stepper motor 28 tomove the plunger 34 down, aspirating a discrete quantity of air (airgap), e.g., 50 microliters in volume into the microdispenser 16. Thecontrol logic 42 then instructs the robotic system 58 to move themicrodispenser 16 down until it makes contact with the surface of thetransfer liquid 24 (not shown) is made. Contact of the microdispenser 16with the surface of the transfer liquid 24 is determined by a capacitiveliquid level sense system (U.S. Pat. No. 5,365,783). The microdispenseris connected by electrical wire 55 to the liquid level sense electronics54. When the liquid level sense electronics 54 detects microdispenser 16contact with transfer liquid 24 surface, a signal is sent to the roboticsystem 58 through electrical wire 53 to stop downward motion.

The control logic 42 next instructs the pump 12 to move the plunger 34down in order to aspirate transfer liquid 24 into the microdispenser 16.The pressure signal is monitored by control logic 42 during theaspiration to ensure that the transfer liquid 24 is being successfullydrawn into the microdispenser 16. If a problem is detected, such as anabnormal drop in pressure due to partial or total blockage of themicrodispenser, the control logic 24 will send a stop movement commandto the pump 12. The control logic 24 will then proceed with an encodedrecovery algorithm. Note that transfer liquid 24 can be drawn into themicrovolume liquid handling system 10 up to the pressure sensor 14without threat of contaminating the pressure sensor 14. Additionaltubing can be added to increase transfer liquid 24 capacity. Once thetransfer liquid 24 has been aspirated into the microdispenser 16, thecontrol logic 42 instructs the robotic system 58 to reposition themicrodispenser 16 above the chosen target, e.g., a microtitre plate.

In one preferred embodiment the microdispenser 16 is the MD-K-130Microdispenser Head manufactured by Microdrop, GmbH, Muhlenweg 143,D-22844 Norderstedt, Germany.

As illustrated in FIG. 3, the microdispenser 16 consists of apiezoceramic tube 60 bonded to a glass capillary 62. The piezoceramictube has an inner electrode 66 and an outer electrode 68 for receivinganalog voltage pulses which cause the piezoceramic tube to constrict.Once the glass capillary 62 has been filled with transfer liquid 24, thecontrol logic 42 directs the microdispenser electronics 51 by electricalwire 50 to send analog voltage pulses to the piezoelectric transducer 60by electrical wire 52. In one preferred embodiment the microdispenserelectronics 51 is the MD-E-201 Drive Electronics manufactured byMicrodrop, GmbH, Muhlenweg 143, D-22844 Norderstedt, Germany. Themicrodispenser electronics 51 control the magnitude and duration of theanalog voltage pulses, and also the frequency at which the pulses aresent to the microdispenser 16. Each voltage pulse causes a constrictionof the piezoelectric transducer 60, which in turn deforms the glasscapillary 62. The deformation of the glass capillary 62 produces apressure wave that propagates through the transfer liquid 24 to themicrodispenser nozzle 63 where one droplet 26 of transfer liquid 24 isemitted under very high acceleration. The size of these droplets 26 hasbeen shown to be very reproducible. The high acceleration of thetransfer liquid 24 minimizes or eliminates problems caused by transferliquid 24 surface tension and viscosity, allowing extremely smalldroplets 26 to be expelled from the nozzle, e.g., as small as 5picoliter droplets 26 have been demonstrated. Use of the microdispenser16 to propel droplets 26 out of the nozzle also avoids problemsencountered in a liquid transfer technique called touchoff. In thetouchoff technique, a droplet 26 is held at the end of the nozzle and isdeposited onto a target surface by bringing that droplet 26 into contactwith the target surface while it is still hanging off of themicrodispenser 16. Such a contact process is made difficult by thesurface tension, viscosity and wetting properties of the microdispenser16 and the target surface which lead to unacceptable volume deviations.The present invention avoids the problems of the contact process becausethe droplets 26 are expelled out of the microdispenser 16 at a velocityof several meters per second. The total desired volume is dispensed bythe present invention by specifying the number of droplets 26 to beexpelled. Because thousands of droplets 26 can be emitted per secondfrom the microdispenser 16, the desired microvolume of transfer liquid24 can rapidly be dispensed.

In one preferred embodiment, the lower section of the glass capillary62, between the piezoelectric transducer 60 and the nozzle 63, is platedwith a conductive material, either platinum or gold. This provides anelectrically conductive path between the microdispenser 16 and theliquid level sense electronics 54. In one preferred embodiment the glasscapillary 62 has an overall length of 73 millimeters, and the nozzle 63has an internal diameter of 75 micrometers.

To dispense microvolume quantities of transfer liquid 24, analog voltagepulses are sent to the microdispenser 16, emitting droplets 26 ofliquid. Capillary forces acting on the transfer liquid 24 replace thevolume of transfer liquid 24 emitted from the microdispenser 16 withliquid from the tubing 18. However, since the transfer liquid-airgap-system liquid column terminates at a closed end in the positivedisplacement pump 12, there is a corresponding drop in the system liquid20 line pressure as the air gap 22 is expanded. This is illustrated inFIG. 4 which depicts the pressure profile measured during amicrodispense of 500 nanoliters. Important to the present invention, themagnitude of the pressure drop is a function of the size of the air gap22 and the volume of the liquid dispensed.

With an air gap 22 of known volume, the pressure change as detected bythe pressure sensor 14 relates to the volume dispensed. Thus, thecontrol logic 42 determines from the pressure change measured by thepressure sensor 14, the volume of transfer liquid 24 that was dispensed.In one preferred embodiment of the present invention it is preferablethat the drop in pressure not exceed approximately 30 to 40 millibarsbelow ambient pressure, depending on the properties of the transferliquid 24. If the amount of transfer liquid 24 dispensed is sufficientto drop the pressure more than 30 to 40 millibars, the pressuredifference across the microdispenser 16, i.e., between the ambientpressure acting on the nozzle 63 and the pressure at the capillary inlet63, will be sufficient to force the transfer liquid 24 up into thetubing 18. This will preclude further dispensing. There is a maximumamount of transfer liquid 24 that can be dispensed before the controllogic 42 is required to command the pump 12 to advance the plunger 34 tocompensate for the pressure drop. This maximum volume is determined bythe desired dispense volume and the size of the air gap 22. Conversely,the size of the air gap 22 can be selected based on the desired dispensevolume so as not to produce a pressure drop exceeding 30 to 40 millibarsbelow ambient pressure. It is also within the scope of the presentinvention to advance the plunger 34 while the microdispenser 16 isdispensing, thereby rebuilding system liquid 20 line pressure, so thatthe microdispenser 16 can operate continuously.

The change in system liquid 20 pressure is used to determine that thedesired amount of transfer liquid 24 was dispensed. A secondverification of the amount of transfer liquid 24 that was dispensed ismade by the control logic 42 monitoring the system liquid 20 linepressure while directing the pump 12 to advance the syringe plunger 34upwards towards Position 1. The syringe plunger 34 is advanced until thesystem liquid 20 line pressure returns to the initial (pre-dispense)value. By the control logic 42 tracking the displaced volume the plunger34 moves (20.83 nanoliters per stepper motor 28 step), a secondconfirmation of dispensed volume is made, adding robustness to thesystem. The system liquid 20 line pressure is now at the correct valuefor the next microdispenser 16 dispense, if a multi-dispense sequencehas been specified.

Once the transfer liquid 24 dispensing has been completed, the controllogic 24 causes the robotic system 58 to position the microdispenser 16over the wash station. The control logic 24 then directs pump 12 androbotic system 58 in a wash sequence that disposes of any transferliquid 24 left in the microdispenser 16, and washes the internal surfaceof glass capillary 62 and the external surface in the nozzle 63 areathat was exposed to transfer liquid 24. The wash fluid can either besystem liquid 20 or any other liquid placed onto the deck of the roboticsystem 58. The wash sequence is designed to minimize cross-contaminationof subsequent transfer liquids 24 with transfer liquids processed prior.Toward this end, it is also possible to enable an ultrasonic wash of themicrodispenser 16. This is accomplished by the control logic 42directing the microdispenser electronics 51 to end electrical pulses tothe microdispenser at a frequency in the ultrasonic range, .g., 12-15kilohertz, that coincides with a resonant frequency of themicrodispenser 16--transfer liquid 24 system.

In the above description of the invention, the control of themicrodispenser 16 was effected by sending a specific number ofelectrical pulses from the microdispenser electronics 51, each producingan emitted droplet 26 of transfer liquid 24. It is also within the scopeof the invention to control the microdispenser 16 by monitoring thepressure sensor 14 signal in realtime, and continuing to send electricalpulses to the microdispenser 16 until a desired change in pressure isreached. In this mode of operation, the PC-LPM-16 Multifunction I/OBoard that contains the A/D converter 44 is instructed by control logic42 to send electrical pulses to the microdispenser electronics 51. Eachpulse sent by the Multifunction I/O Board results in one electricalpulse that is sent by the microdispenser electronics 51 to themicrodispenser 16, emitting one droplet 26 of transfer liquid 24. Thecontrol logic 42 monitors the pressure sensor 14 signal as themicrodispenser 16 dispense is in progress, and once the desired changeis pressure has been attained, the control logic 42 directs theMultifinction I/O Board to stop sending electrical pulses.

This mode of operation is employed if a "misfiring" of microdispenser 16has been detected by control logic 42.

It is also within the scope of the invention for the microvolume liquidhandling system 10 to automatically determine (calibrate) the size ofthe emitted droplets 26 for transfer liquids 24 of varying properties.As heretofore mentioned, emitted droplet 26 size is affected by theproperties of the transfer liquid 24. Therefore, it is desirable to beable to automatically determine emitted droplet 26 size so that the userneed only specify the total transfer volume, and the system 10 willinternally determine the number of emitted droplets 26 required tosatisfy the user request. In the encoded autocalibration algorithm, oncethe system 10 is primed, an air gap 22 and transfer liquid 24 aspirated,the control logic 42 instructs microdispenser electronics 51 to send aspecific number of electrical pulses, e.g., 1000, to the microdispenser16. The resulting drop in pressure sensor 14 signal is used by controllogic 42 to determine the volume of transfer liquid 24 that wasdispensed. This dispensed volume determination is verified by thecontrol logic 42 tracking the volume displaced by the movement of theplunger 34 to restore the system liquid 20 line pressure to thepre-dispense value.

The microvolume liquid handling system 10 illustrated is FIG. 1 depictsa single microdispenser 16, pressure sensor 14, and pump 12. It iswithin the spirit and scope of this invention to include embodiments ofmicrovolume liquid handling systems that have a multiplicity (e.g., 4,8, 96) of microdispensers 16, pressure sensors 14, and pumps 12. It isalso within the spirit and scope of this invention to includeembodiments of microvolume liquid handling systems that have amultiplicity of microdispensers 16, pressure sensors 14, valves 38, andone or more pumps 12.

What is claimed is:
 1. A method for dispensing a microvolume of a sampleliquid and determining the volume of the sample liquid that wasdispensed from a closed container including a dispenser with a passagetherethrough, said passage having a tip portion with a tip opening, saidmethod comprising the following steps:(a) supplying a system liquid intothe closed container; (b) supplying the sample liquid into at least thetip portion of the passage; (c) introducing a compressible fluid intosaid closed container between the system liquid and the sample liquid,such that the compressible fluid separates said system liquid from saidsample liquid; (d) dispensing a portion of said sample liquid from saidcontainer through said tip opening, without dispensing said compressiblefluid; and, (e) determining the microvolume of the dispensed sampleliquid based on a change in a parameter in said closed container,resulting from dispensing of said sample liquid from said closedcontainer, said change being responsive to said compressible fluid. 2.The method of claim 1 wherein the step of determining comprises thefollowing steps:determining a pressure change in said closed containerresulting from said dispensing of a microvolume of the sample liquid;and converting said pressure change into the microvolume of the sampledispensed.
 3. The method of claim 2 wherein the determining stepcomprises using a piezoresistive pressure sensor.
 4. The method of claim2 wherein the pressure change is determined at the location of thecompressible fluid.
 5. The method of claim 2 wherein the pressure changeis about 4 millibars.
 6. The method of claim 5 wherein the pressurechange is determined in about one second.
 7. The method of claim 1wherein the step of dispensing comprises:restricting the volume of thepassage through said dispenser for a time period and in the amountsufficient to eject a droplet of sample liquid from said dispenser;allowing the volume to return to its unrestricted state; repeating therestricting and the allowing step to dispense a microvolume of saidsample liquid.
 8. The method of claim 7 wherein each droplet is in therange from 5 picoliters to about 0.45 nanoliters.
 9. The method of claim8 wherein the sample liquid contains one or more biologically activesubstances.
 10. The method of claim 8 wherein the sample liquid containsone or more chemically active substances.
 11. The method of claim 8wherein the sample liquid and the system liquid are the same.
 12. Amethod for dispensing a microvolume of a sample liquid and determiningthe volume of the sample liquid that was dispensed from a closedcontainer including a dispenser with a passage therethrough, saidpassage having a tip portion with a tip opening, said method comprisingthe following steps:(a) supplying the system liquid into the closedcontainer; (b) supplying the sample liquid into at least the tip portionof the passage; (c) introducing a compressible gas into said closedcontainer between the system liquid and the sample liquid, said gasseparating said system liquid from said sample liquid; (d) repetitirelyconstricting the volume of said passage to dispense in the form ofdroplets through said tip opening, without dispensing said gas, aportion of said sample liquid to produce the microvolume of sampleliquid;determining a pressure change in said closed container resultingfrom said dispensing of a microvolume of the sample liquid; and,converting said pressure change into the microvolume of the sampledispensed.
 13. The method of claim 12 wherein the determining stepcomprises using a piezoresistive pressure sensor.
 14. The method ofclaim 12 wherein the pressure change is determined at the location ofthe compressible gas.
 15. The method of claim 12 wherein the pressurechange is about 4 millibars.
 16. The method of claim 15 wherein thepressure change is determined in about one second.
 17. The method ofclaim 12 wherein each droplet is in the range from 5 picoliters to about0.45 nanoliters.
 18. The method of claim 17 wherein the sample liquidcontains one or more biologically active substances.
 19. The method ofclaim 17 wherein the sample liquid contains one or more chemicallyactive substances.
 20. The method of claim 12 wherein the sample liquidand the system liquid are the same.
 21. The method of claim 12 whereinthe constricting step is carried out using a piezoelectric element. 22.A method for dispensing a microvolume of a sample liquid and determiningthe volume of the sample liquid that was dispensed from a closedcontainer including a capillary with a passage therethrough, saidpassage having a tip portion with a tip opening, said method comprisingthe following steps:(a) supplying the system liquid into the closedcontainer; (b) supplying the sample liquid into at least the tip portionof the passage;introducing compressible gas into said closed containerbetween the system liquid and the sample liquid, said gas separatingsaid system liquid from said sample liquid; restricting the volume ofsaid passage for a time period and in the amount sufficient to eject adroplet of a sample liquid from said tip opening, without dispensingsaid gas; allowing the volume of said passage to return to itsunrestricted state; repeating the restricting and the allowing steps todispense a portion of said sample liquid without dispensing said gas,said portion being the microvolume of said sample liquid; determining apressure change in said closed container resulting from said dispensingof said microvolume of the sample liquid; and converting said pressurechange into the microvolume of the dispensed sample.
 23. The method ofclaim 22 wherein the determining step comprises using a piezoresistivepressure sensor.
 24. The method of claim 22 wherein the pressure changeis determined at the location of the compressible gas.
 25. The method ofclaim 24 wherein the pressure change is determined in about one second.26. The method of claim 25 wherein the sample liquid contains one ormore biologically active substances.
 27. The method of claim 25 whereinthe sample liquid contains one or more chemically active substances. 28.The method of claim 22 wherein the pressure change is about 4 millibars.29. The method of claim 22 wherein each droplet is in the range from 5picoliters to about 0.45 nanoliters.
 30. The method of claim 22 whereinthe sample liquid and the system liquid are the same.
 31. The method ofclaim 22 wherein the restricting step is effected using a piezoelectricelement.